TMC-95A–D et Analogues : Design et Synthèse de Nouveaux Inhibiteurs du Protéasome à Visée Anti-tumorale – ProteInh

Cast aside in recent years, natural product drug discovery is now making a comeback, for three reasons. Firstly, combinatorial chemistry's promise to fill drug development pipelines with new synthetic small-molecule drug candidates is as yet unfulfilled. Secondly, the practical difficulties of natural product drug discovery are being overcome by advances in separation technologies and in the speed and sensitivity of structure elucidation. Thirdly, a compelling case can be made for the intrinsic utility of natural products as sources of drug leads. A glance at the figures is simply astounding: 61% of the 877 small-molecule new chemical entities introduced as drugs worldwide from 1981 to 2002 can be traced to or were inspired by natural products. The research project that we propose was developed from this perspective, based on the structure of natural products with promising reversible, non-covalent proteasome inhibition properties: the TMC-95A–D. Realization of the proteasome's importance in various aspects of cell biology has prompted increased efforts in the field of proteasome inhibition since selective inhibitors offer an amazing therapeutic potential and now clearly appear as excellent targets for cancer chemotherapy. Drug resistance and lack of tumor specificity frequently hinder the treatment of neoplastic disease, creating a need for new classes of potent and specific anticancer drugs. In preclinical cancer models, proteasome inhibitors have been shown to induce potently apoptosis in many types of cancer cells, with reduced cytotoxicity in normal cells. They have also been shown to have in vivo antitumor efficiency. Therefore, proteasome inhibition holds promise as a novel approach to the treatment of cancer, as demonstrated with Velcade®, the first such inhibitor to be approved for therapy, a compound that however displays too many side-effects due to its poor specificity. To increase the potency and therapeutic utility of proteasome inhibitors, one must improve their specificity. As TMC-95A–D have a unique mode of action and block the active sites of the proteasome non-covalently by a strong array of hydrogen bonds, their structure clearly constitutes a new lead for the design of specific proteasome inhibitors with increased activity in the tumor-targeting approach. The project is set out in three stages: development of a general synthetic pathway to the TMC-95A–D, rational design and synthesis of analogues, and evaluation of their potency as specific proteasome inhibitors together with their ability to reduce cancer cell proliferation and mobility. The first phase of this project, the development of an original, efficient and simple synthetic method, is based on the expertise of the team in the field of peptide chemistry and synthesis of complex cyclopeptides. Importantly, emphasis has been placed on the creation of a procedure which will allow multiple alternative routes to complete the syntheses successfully. We propose a novel synthetic pathway, which brings together peptide coupling steps with recent synthetic methodology; the use of this synthetic path and the reactions within it will be validated by the synthesis of TMC-95A. Next, we will concentrate on the synthesis of analogues and their rational optimization in a tumor-targeting approach. For this we propose to begin with modifications of the side chains of the natural product, which are responsible for binding in the S1 and S3 pockets of active sites and provide most of the specific interactions. This should result in analogues that would possess a greater specificity for the different proteasome's subunit, and we will target the beta5 site responsible for CT-L activity associated with tumor cell survival. In parallel, a second library will be created by adding to the cyclopeptide structure a 'warhead' capable of reacting with the catalytic threonine. The combination of the macrocyclic structure possessing a high affinity for the active site and this warhead capable of reacting with it could provide a certain level of synergy and therefore result in an increase in biological activity. For this stage of the project, the use of modelization of the analogues within the active site should ease and expedite their design. Finally, members of these two libraries will be evaluated for their potency as specific proteasome inhibitors (evaluation of their chymotrypsin like, trypsin like and post glutamyl peptide hydrolyzing inhibitory activities) together with their ability to reduce cancer cell proliferation and mobility. At this final stage of the project, comparison of the biological activities of the members of the two libraries will allow the establishment of a more complete structure-activity relationship profile, and to determine the optimal structure.